The invention relates to a miniaturized temperature-zone flow reactor used for thermally controlled biochemical and molecular-biological respectively, in particular for the method of the so-called polymerase chain reaction, referred to as PCR in the following, in which definite sequences out of a mixture of DNA-sequences are amplified.
When carrying out thermally controlled biochemical and molecular-biologic processes, respectively, very often different temperatures are required to be applied to the processing steps. Such an applying of changing temperatures are of particular importance with the so-called PCR.
The method of PCR has been developed in the recent years for amplifying definite DNA-sequences, and its principles have been specified by Darnell, J.; Lodish, H.; Baltimore, D. in “Molekular Zellbiologie, Walter de Gruyter, Berlin-New York 1994, p. 256/257”. It is an essential, inter alia, with said method that mixtures of DNA-sequences are subjected to a definite changing temperature treatment. Thereby stationary sample treatment equipment is used, in which the respective samples are filled into sample chambers and are then periodically subjected to a heat-cold temperature cyclic treatment in the course of which, depending on the definitely pre-set primers, the desired DNA-sequences are amplified. In this respect, the effectiveness of the sample chambers known up to now is considered as not being sufficient. For this reason there has recently been proposed a miniaturized sample chamber (Northrup et al, DNA Amplification with microfabricated reaction chamber, 7th International Conference on Solid State Sensors and Actuators, Transducers Proceedings 93 (June 1993, Yokohama, Japan), p. 924-26), which permits a four times faster amplification of the desired DNA-sequences compared to the prior arrangements. This sample chamber, which can take up to 50 ul sample fluid, consists of a structurized silicon cell having a longitudinal extension in an order of size of 10 mm which is closed, in a sample affecting direction, by a thin membrane, to which the corresponding temperature is applied by way of a miniaturized heating element. Also with this device the DNA-sequences to be amplified are inserted via micro-channels into the chamber, then they are subjected to a polymerase chain reaction, and subsequently removed. In spite of the advantages obtained by this device, it involves substantially the disadvantage that also this sample chamber has to be heated and cooled as a whole which only permits limited rates of temperature changes. Particularly, when further reducing the size of the sample, the parasitic heat capacity of the sample chamber and that of a necessary temper block, if any, becomes more weighty compared to the sample fluid, so that the possible high temperature cycle rates which are attainable, in principle, with small liquid volumes cannot be obtained, whereby the effectiveness of the method is comparatively low. Moreover, a comparatively expensive control system is necessary each time a constant temperature schedule for a same fluid is to be obtained, and the heating and cooling power provided are substantially consumed in the components surrounding the sample fluid rather than in the sample fluid itself.
Furthermore, there is known from U.S. Pat. No. 5,270,183 a thermo-cycler operating on the flow principle in which the sample fluid to be amplified is passed through a pipe that sequentially is once or multiply wound around a plurality of cylinders which are kept at different temperatures. Such an embodiment, in principle, permits an amplification of comparatively small amounts of samples, down to about 25 μl. The manipulation of such a device, however, is rather impracticable and requires a high production skill in the manufacture of such devices, so that they are completely unsuited for serial production.
A flow thermo-cycler described in WO96/10456, comes nearest to the present invention, in which structurizing technologies known from the so-called micro-system technology are used to provide a sample receiving chamber. This sample receiving chamber permits a dynamic sample treatment of even very small amounts of very expensive substances. The achievement of this proposed solution is that the sample partial volumes are subjected to a homogeneous temperature throughput in respectively provided heating and cooling zones, also resulting in an increase of the output with respect to the samples to be amplified. Furthermore, and due to the design depending omission of the heating and cooling procedures for the wall materials and the severe minimizing of the parasitic heat capacities and heat influences, not only the required expenditures for the control are considerably lower, but also the entire cycle of the process is substantially time-reduced. Thereby only as much heating and cooling power has to be fed in as can be transported in the stream of the sample fluid. Additionally, the embodiment of the thermo-cycler described in WO 96/10456 not only permits a continuous process control, but also a serial operation in which different substances can be sequentially introduced into the thermocycler without the danger of an interfering mixing with the sample which is still in the device. This solution, however, is disadvantageous because, on the one hand, there is required a very precise, structured procedure for manufacturing the membranes provided therein, and, on the other hand, due to the set-up of the device described there, the retention time of the sample fluid in the cooling zone ranges is undesiredly high, at least at partial passages, which can lead to the formation of undesired by-products when carrying out PCR.